PROJECT SUMMARY/ABSTRACT This proposal addresses the problem of bone healing in large segmental osseous defects that occur as a result of injury, tumor resection or the correction of congenital deformities. They present important clinical problems because they heal poorly and there are no clinically expedient ways to regenerate the lost bone. Much interest was aroused by the cloning of bone morphogenetic proteins (BMPs), which have potent osteogenic properties in animal models. Recombinant BMP-2 and BMP-7 advanced to clinical use, but they have improved the field only incrementally. Their disappointing clinical performance is thought to reflect delivery problems. The proteins are mixed with a collagen scaffold and surgically implanted into a bone defect in the operating room. Most of the BMP leaves the defect within hours. To address this, massively supraphysiological amounts of the BMP are implanted, leading to major side-effects and greatly increasing the cost. Attempts to improve the delivery of the BMPs have focused on the development of scaffolds that incorporate BMPs and release them slowly after implantation, and gene therapy, which enables cells within and around the osseous defect to synthesize their own BMP endogenously. Recent data from the latter approach demonstrate that, when the BMP is synthesized endogenously, expression of the BMP needs to be neither prolonged nor high for effective bone healing. This raises the possibility of delivery mRNA rather than DNA to enable local, endogenous synthesis of the BMP. While theoretically satisfying, this approach is restricted because mRNA is unstable, toxic to cells and inflammatory. This project will explore the use of chemically modified RNA (cmRNA) that lacks these disadvantages. Among the chemical modifications are the inclusion of iodo-substituted pyrimidines uracil and cytosine; the introduction of specific 5?-untranslated regions (UTRs); and the inclusion of an extended polyA tail. Chemically modified RNA technology was invented and pioneered at the Technical University of Munich (TUM), which has produced a cmRNA encoding BMP-2. Pilot studies in the PI?s laboratory in collaboration with TUM have confirmed that BMP-2-cmRNA heals critical size defects in the rat femur with impressive speed and reliability. Funds are thus requested by this collaboration to develop the work further with the goal of eventual human trials. This proposal seeks to establish the central premise that healing of critical size defects is effective when BMP-2 is expressed locally and transiently from cmRNA molecules. A rat, femoral critical sized defect will be used in both male and female animals. Specific Aim 1 will determine the location, duration and level of cmRNA expression. Specific Aim 2 will study the biology of healing, particularly with regard to identifying the cells that express and respond to BMP-2. Because large animal studies are a necessary prelude to contemplating human trials, Specific Aim 3 will use a sheep drill hole model to establish whether BMP-2-cmRNA is osteogenic in sheep.